CN114181871B - Brevibacillus laterosporus strain and application thereof - Google Patents

Brevibacillus laterosporus strain and application thereof Download PDF

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CN114181871B
CN114181871B CN202111680045.4A CN202111680045A CN114181871B CN 114181871 B CN114181871 B CN 114181871B CN 202111680045 A CN202111680045 A CN 202111680045A CN 114181871 B CN114181871 B CN 114181871B
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brevibacillus laterosporus
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fermentation broth
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CN114181871A (en
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麻林涛
王莉
王付勇
张恒
田晓
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Henan Dr Ma Xiwanjia Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—Microorganisms; Enzymes; Antibiotics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • Health & Medical Sciences (AREA)
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  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Soil Sciences (AREA)
  • Environmental & Geological Engineering (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a strain of Brevibacillus laterosporus (Brevibacillus laterosporus) and application thereof, which relate to a microorganism application technology, wherein the collection number of the strain is CGMCC No.22964, and fermentation products of the strain are outstanding in resisting botrytis cinerea and degrading carbamate pesticide pollution.

Description

Brevibacillus laterosporus strain and application thereof
Technical Field
The invention relates to a microorganism application technology, in particular to a strain containing specific Brevibacillus laterosporus (Brevibacillus laterosporus) and application thereof.
Background
Brevibacillus laterosporus (Brevibacillus laterosporus) is an aerobic bacterium which is distributed in cold zone, warm zone and tropical zone and exists on plant surface, in insect body, in soil, in fresh water or in sea water. Various metabolites with different levels can be produced in the growth process of the bacillus laterosporus thallus, such as bacillus subtilis amine, peptide antibiotics, polyketide, chitinase and the like, and some excellent strains are prepared into bacteriostats, immunomodulators, soil improvers and the like according to the characteristics of the excellent strains and are widely applied to the fields of medicines, agriculture, and aquaculture.
The quality and the efficacy of the Brevibacillus laterosporus preparation product mainly depend on the characteristics of strains, and good strains are screened out to provide a premise of high-quality products. Degradation, variation and the like of the strain can occur in the passage process; therefore, there is a need to continuously develop and screen excellent strains to provide candidate materials for Brevibacillus laterosporus preparation products.
Disclosure of Invention
In order to enrich the available products in the field and provide a high-quality agricultural biocontrol microbial inoculum, the invention develops and screens to obtain a multifunctional Brevibacillus laterosporus strain, which is specifically as follows:
a strain CB202103-8 of Brevibacillus laterosporus (Brevibacillus laterosporus) has a preservation number of CGMCC No.22964.
A fermentation broth containing the above Brevibacillus laterosporus.
The application of the Brevibacillus laterosporus in resisting the gray mold of tomatoes is characterized by comprising any one of the following measures
(1) Spraying the fermentation liquor on tomato plants at any stage of the growth and development period of the tomatoes;
(2) Spraying the fermentation liquor on picked tomatoes;
(3) Spraying the fermentation liquor on the soil of the tomato cultivation land;
the effective concentration of the Brevibacillus laterosporus in the fermentation liquid is 1 multiplied by 10 5 ～1×10 7 CFU/mL; or 5X 10 5 ～ 5×10 6 CFU/mL.
The application of the Brevibacillus laterosporus in preparing soil conditioners.
The application of the Brevibacillus laterosporus in treating soil organic pesticide pollution is characterized in that the fermentation liquor of the Brevibacillus laterosporus is sprayed into the farmland to be treated.
According to the invention, 150 strains separated from farmland environment are subjected to screening for resisting tomato gray mold pathogenic bacteria and carbamate degradation test, a strain of Brevibacillus laterosporus (Brevibacillus laterosporus) is selected, and the strain can resist tomato gray mold pathogenic bacteria and greatly degrade carbamate pesticides contained in soil through test detection. The microbial inoculum is beneficial to preventing and controlling the gray mold of tomatoes, improving the yield and the quality of tomatoes and reducing the loss; the isolated strain is sent to China general microbiological culture Collection center for preservation, and the following technical scheme is claimed:
preservation information:
preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation address: beijing city, chaoyang area, north Chenxi lu 1, 3, 100101
Preservation date: 2021, 7, 27
The strain names are as follows: CB202103-8
The preservation number is: CGMCC No.22964.
Classification naming: brevibacillus laterosporus Brevibacillus laterosporus
Detailed Description
The invention is illustrated by the following specific examples.
1. Isolation of strains
Sampling time: 2021 month 3
Sampling site: zhengzhou Henan
Sampling 150 parts of farmland soil, 5-10g of each part of soil, filling each part of soil into a triangular flask, adding 200ml of sterilized water, stirring uniformly, oscillating for 10 minutes to prepare suspension, taking 1ml of the suspension, adding 9ml of double distilled water, and carrying out gradient dilution by using the double distilled water; and (3) coating 200ul of diluent on an LB plate culture medium, culturing for 20-50 hours at 25-35 ℃, picking up dominant single colonies with consistent forms, transferring to the LB plate culture medium for streaking and purification, taking part for analysis, detection and screening, and storing the rest in a refrigerator at-70 ℃.
2. Strain screening and identification
2.1 screening of Botrytis cinerea pathogenic bacteria antagonistic bacteria
Botrytis cinerea (CGMCC No. 3.4583) is purchased from China general microbiological culture Collection center.
Culture medium: PDA medium: 200g of potato, 20g of glucose, 15-20g of agar, 1 000mL of tap water and natural pH; PDB liquid medium: agar was not added and other ingredients were the same as PDA medium.
The pathogenic bacteria were inoculated on PDA medium and cultured upside down at 22 ℃.
A flat plate facing method is adopted. Activated pathogenic bacteria cake (diameter of 6 mm) is placed in the center of PDA plate, and cultured at 22deg.C for 24 hr; the purified strain to be detected is picked up by a sterilized toothpick and inoculated on 4 corner points 3cm away from the center of a plate, and is cultured for 3-5d at 30 ℃.3 repeats are set for each treatment, a plate inoculated with pathogenic bacteria is used as a control, the growth state of the pathogenic bacteria is observed, a bacterial strain which has an inhibition effect on the growth of the pathogenic bacteria and has a flush edge of the inhibitory mycelium and a durable antagonism is selected, the colony diameter of the pathogenic bacteria is measured, and the inhibition rate of the bacterial strain is calculated:
inhibition (%) = [ (control colony diameter-treatment colony diameter)/(control colony diameter-original cake diameter) ] x 100%.
In the experiment, 20 candidate strains are selected, the inhibition rate is 65.05-80.31%, and the inhibition rate of CB202103-8 claimed by the invention reaches 78.85%.
2.2 identification of antagonistic bacteria morphology
And carrying out morphological identification on the selected candidate antagonistic strains.
On LB culture medium, CB202103-8 disclosed by the invention has smooth colony edge, is opaque and grey white, and has no wrinkles on the surface. Observation under electron microscope: the bacterial rods, the periphyton and the colony have the size of about (0.6-1) mu m x (2.0-4.0) mu m, and the spores are visible on the side.
Gram staining results were variable: delay period G - Log phase G + At the stationary phase G - 。
The selected strain CB202103-8 is detected by using a Phoenix (TM) 100 type full-automatic microorganism analyzer for identification and referring to a strain physiological and biochemical detection method of the Burger bacteria identification handbook:
the strain CB202103-8 can normally grow in the range of 10-40 ℃ and the pH value of 7-9, and the optimal growth temperature is 20-30 ℃ and the pH value of 7.2-8.0. The preliminary identification is determined to be Brevibacillus laterosporus.
2.3 16S rDNA sequence analysis and identification
The 16S rDNA sequence amplification specific primers were as follows, for the delegated synthesis:
forward Primer A5'-AGAGTTTGATCCTGGCTCAG-3' (Seq ID No. 1),
reverse Primer B:5'-TACGGATACCTTGTTACGACTT-3' (Seq ID No. 2).
By TaKaRa TM The bacterial genome DNA extraction kit extracts genome DNA of candidate strains, and PCR amplification is performed by taking the extracted genome DNA as a template.
PCR reaction System (50. Mu.L): taKaRa LA-Taq 0.5. Mu.L, DNA template 2. Mu.L, 10 XPCR Buffer 5. Mu.L, dNTP mix 8. Mu.L, upstream and downstream primers 2. Mu.L each, double distilled water 30.5. Mu.L.
PCR procedure: pre-denaturation at 94℃for 5min,94℃for 30s,54℃for 1min,72℃for 2min,30 cycles; extending at 72℃for 7min.
After the PCR products are purified, sequencing is carried out, and the sequencing results obtained are subjected to Blast comparison analysis through NCBI database.
The results show that: the 16S rDNA fragment of the strain CB202103-8 selected by the invention has the size of 1526bp, and the similarity is 99% when compared with the known Brevibacillus laterosporus.
The evolution relationship is analyzed by MEGA5.0 software, and the result shows that CB202103-8 has the closest relationship with Brevibacillus laterosporus and is determined to be Brevibacillus laterosporus.
The isolated strain is sent to China general microbiological culture Collection center for preservation, and the name of the strain is: CB202103-8, accession number: CGMCC No.22964.
3. Strain detection
3.1 Brevibacillus laterosporus CB202103-8 control effect detection on tomato gray mold
Activating the preserved CB202103-8 strain on an LB culture medium for 24 hours, picking single bacterial colonies, and inoculating the single bacterial colonies into 20mL of LB liquid culture medium to obtain strain liquid. Then inoculating 1% of the strain into 100ml of culture medium to culture at 37 ℃ and 200r/min constant temperature shaking culture for 24 hours to obtain CB202103-8 culture solution. Dilution with sterile Water to 1X10 7 CFU/mL。
Punching the edge of Botrytis cinerea growing on PAD culture medium for 5 days with a puncher, inoculating the bacterial block into 50mL sterile water, shake culturing at 28deg.C and 180rpm for 10 min, and diluting to 1×10 6 CFU/mL。
Sterilizing the surfaces of tomato fruits: wiping with 75% alcohol for 30s, and cleaning with sterile water for 3 times. Punching holes on the surface of each fruit by using a sterilizing gun head, wherein 6 holes are formed on the surface of each fruit; 10 fruits were treated each. The concentration of the culture solution of the treatment group CB202103-8 is 1 multiplied by 10 respectively 7 、 5×10 6 、1×10 6 、5×10 5 And 1X10 5 CFU/mL, 20. Mu.L of culture solution was added to each well, and after 2 hours, 20. Mu.L of 1X10 was added 6 The CFU/mL of the botrytis cinerea suspension is inoculated with only botrytis cinerea as a positive control CK1 and sterile water as a negative control CK2. The control effect of antagonistic bacterium CB202103-8 on tomato gray mold (Botrytis cinerea) is measured, the tomato gray mold is subjected to dark and moisture preservation for 48 hours, the tomato gray mold is subjected to constant temperature culture at 28 ℃, the disease condition is observed, and the disease rate and the disease index are calculated.
Disease severity grading criteria were as follows: grade 0 no disease; 1 grade 1 disease hole; 2 stage 2 disease wells; 3 stage 3 disease holes; 4 grade 4 disease wells; grade 5-6 disease wells.
Disease index (%) = { Σ [ (number of disease fruits at each stage×relative number) ]/investigation of total fruits×5} ×100%
Control effect (%) = { [ positive control disease index-treated disease index ]/positive control disease index } ×100%
The results of the investigation are shown in Table 1 below:
TABLE 1 control of CB202103-8 on Botrytis cinerea
As can be seen from Table 1, tomatoes added with high concentration of antagonistic bacterium CB202103-8 do not develop disease at day 3, reach 100%, 88.2% at day 5, and still reach 80.5% at day 7; when the antagonistic concentration is reduced to 5×10 6 At CFU/mL, the control effect is not remarkably different from the control effect at the 3 rd and 5 th days, but the control effect is obviously reduced at the 7 th day, which indicates that the control effect duration of the low concentration is reduced; when the concentration of antagonistic bacteria A60 is lower than 5×10 5 The CFU/mL is reduced, but the CFU/mL has obvious prevention effect. As a biological antibacterial agent, the use concentration is recommended to be 1 multiplied by 10 5 ～1×10 7 CFU/mBetween L, preferably 5X 10 5 ～ 5×10 6 CFU/mL.
3.2 test of adsorption and degradation Functions of soil carbamate pesticides
The prior art reports that Brevibacillus laterosporus can degrade organic pollutants in soil. The carbamate compounds are widely used as pesticides, acaricides, herbicides [ benazolin, N- (3, 4-dichlorophenyl) methyl carbamate) ] and bactericides on agricultural chemicals, so that carbamate pesticides are accumulated in large-area farmlands, pesticide residues in vegetables and fruits are caused, and the carbamate pesticides are key detection varieties of pesticide residues in current vegetables.
Therefore, the invention carries out carbamate pesticide degradation test on 20 candidate antagonistic strains (comprising CB 202103-8) selected in 2.1, and hopefully selects dominant strains with better tomato gray mold resistance and organic pollutant degradation functions.
Reagent:
"Miao Cao Ling" (CAS#: 1918-18-9), 0.1Mg/ML, beijing Bao Ling technology Co., ltd
Experiment design:
experimental group 1. In 250mL triangular flask with plug, 100g soil sample is added, and 5×10 culture solution of antagonistic strain LB medium is added 6 CFU/mL 5mL, then 100mL (concentration 100 ug/Kg) of sterile water diluted benazolin was added; then placing the mixture in a water bath constant temperature oscillator at 25 ℃ for culture, sampling at different time points, centrifuging at a rotating speed of 4,000 g for 20 minutes, and taking the supernatant to measure the concentration of the benazolin;
experimental group 2. In 250mL triangular flask with plug, 5×10 culture solution of antagonistic strain LB medium is added 6 CFU/mL 5mL, then 100mL (concentration 100 ug/L) of sterile water diluted benazolin was added; then placing the mixture in a 28 ℃ water bath constant temperature oscillator for culture, sampling at different time points, centrifuging at a rotating speed of 4,000 g for 20 minutes, and taking the supernatant to measure the concentration of the benazolin;
control group: 100g of soil sample is added into a 250mL triangle bottle with a stopper, and 100mL (the concentration is 100 ug/Kg) of the benazolin diluted by sterile water is added; then placing the mixture in a water bath constant temperature oscillator at 25 ℃ for culture, sampling at different time points, centrifuging at a rotating speed of 4,000 g for 20 minutes, and taking the supernatant to measure the concentration of the benazolin;
the determination of the concentration of benazolin was performed exactly according to the standard procedure of appendix H of GB 5085.6-2007. The measurement results are shown in Table 2.
TABLE 2 dynamic changes in the concentration of benazolin in CB202103-8 treatment
The measurement results show that the experimental groups 1,2 and the control group reach equilibrium after 24 hours; the concentration of the benazolin in the experimental group 1 is reduced by 44% compared with the initial concentration in 12 hours, and is reduced by more than 65% when the equilibrium is reached; the results for experimental group 2 without soil were close to experimental group 1. Indicating that the reduction of the concentration of the benazolin is mainly biologically degraded by the added antagonistic bacteria (CB 202103-8) rather than adsorbed by the soil; the control group also decreased in concentration over time, due to its natural volatilization and degradation; in general, the strain CB202103-8 shows remarkable efficacy in degrading carbamate pesticides represented by benazolin, and is an excellent candidate strain as a soil pollution improver.

Claims (5)

1. A strain CB202103-8 of Brevibacillus laterosporus (Brevibacillus laterosporus) has a preservation number of CGMCC No.22964.
2. A fermentation broth comprising Brevibacillus laterosporus according to claim 1.
3. Use of Brevibacillus laterosporus according to claim 1 for resistance to botrytis cinerea, characterized in that it comprises any one of the following measures:
(1) Spraying the fermentation broth of claim 2 onto tomato plants during the growth and development period of tomatoes;
(2) Spraying the fermentation broth of claim 2 onto the picked tomatoes;
(3) Spraying the fermentation broth of claim 2 on the soil of a tomato cultivation land;
the effective concentration of the Brevibacillus laterosporus in the fermentation liquor is 1 multiplied by 10 5 ～1×10 7 CFU/mL;
the pathogenic bacteria of the tomato gray mold are Botrytis cinerea.
4. The use according to claim 3, wherein the fermentation broth has an effective concentration of Brevibacillus laterosporus of 5X 10 5 ～5×10 6 CFU/mL.
5. The use of Brevibacillus laterosporus according to claim 1 for treating soil organic pesticide pollution, wherein the fermentation liquid of Brevibacillus laterosporus is sprayed into the farm to be treated, and the soil organic pesticide is benazolin.
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Publication number Priority date Publication date Assignee Title
CN104480046A (en) * 2014-12-18 2015-04-01 江苏省农业科学院 Brevibacillus laterosporus strain and application thereof
WO2015167809A1 (en) * 2014-04-28 2015-11-05 Syngenta Participations Ag Method for controlling nematode pests
CN107151641A (en) * 2017-07-04 2017-09-12 沈阳农业大学 One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application
CN111269865A (en) * 2020-04-01 2020-06-12 北京工商大学 Brevibacillus laterosporus strain S62-9 and application thereof
CN111394272A (en) * 2019-12-30 2020-07-10 武汉合缘绿色生物股份有限公司 Brevibacillus laterosporus and application thereof
CN111748496A (en) * 2020-07-03 2020-10-09 天津坤禾生物科技集团股份有限公司 Application of Brevibacillus laterosporus MES818 in tomato cultivation

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WO2015167809A1 (en) * 2014-04-28 2015-11-05 Syngenta Participations Ag Method for controlling nematode pests
CN104480046A (en) * 2014-12-18 2015-04-01 江苏省农业科学院 Brevibacillus laterosporus strain and application thereof
CN107151641A (en) * 2017-07-04 2017-09-12 沈阳农业大学 One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application
CN111394272A (en) * 2019-12-30 2020-07-10 武汉合缘绿色生物股份有限公司 Brevibacillus laterosporus and application thereof
CN111269865A (en) * 2020-04-01 2020-06-12 北京工商大学 Brevibacillus laterosporus strain S62-9 and application thereof
CN111748496A (en) * 2020-07-03 2020-10-09 天津坤禾生物科技集团股份有限公司 Application of Brevibacillus laterosporus MES818 in tomato cultivation

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Title
不同浓度侧孢短芽孢杆菌对土壤与作物生长的影响;苗素平;《济宁学院学报》;第37卷(第6期);第38-42页 *

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